ABSTRACT Interannual variation of Perkinsus olseni infection
intensity in the adult Manila clam Ruditapes philippinarum in Gomso Bay,
off the west coast of Korea, was monitored from 1999 to 2000. Infection
intensity of P. olseni (i.e., total number of P. olseni cells in unit
tissue weight) was determined using Ray's fluid thioglycollate
medium assay and Choi's 2-M NaOH digestion. In Gomso Bay, P. olseni
monthly infection prevalence ranged from 83 (April 2000)--100%. It was
remarkable that of the 18 mo of sampling, the prevalence remained at
100% in 12 sampling months. Infection intensity of P. olseni in Manila
clam ranged 366,001 (July 1999)-2,235,325 cells/g wet tissue (October
1999). The infection intensity recorded in 1999 was significantly higher
than the level measured in 2000, suggesting an interannual variation in
the intensity (P < 0.05). A very high level of infection intensity
observed during the fall (September, October, and November) coincided
with a relatively low condition index and mass morality of clams in the
bay. Our data suggest that the mass mortality of clams observed during
late summer to mid fall in Gomso Bay could be, in part, explained by the
high level of Perkinsus infection coupled with the poor physiological
condition of clams during the postspawning season.

A high level of P. olseni infection in Manila clam has been
reported in Gomso Bay, located off the west coast of Korea, during late
summer. According to Park et al. (1999), the infection intensity of P.
olseni in 2-3-y-old adult Manila clams was 16,777-4,091,667 cells/g
tissue (mean, 1,077,628 cells/g tissue). Such a heavy infection was also
observed in some clam culture grounds on the west and south coast of
Korea (Choi & Park 1997, Park & Choi 2001), although temporal
variation of the infection intensity and prevalence is poorly
investigated (Park et al. 2006a).

In an attempt to understand seasonal dynamics of P. olseni
infection in the Manila clam, we surveyed the infection intensity of P.
olseni in the Manila clam for a 2 y using Ray's fluid
thioglycollate medium assay (RFTM) and the 2-M NaOH digestion technique.
The number of P. olseni cells in whole clam tissue was determined using
RFTM and the 2-M NaOH assay, and the current study reports the annual
variation of P. olseni infection intensity and the prevalence in Manila
clam in Gomso Bay.

MATERIALS AND METHODS

Gomso Bay, located on the west coast of Korea (Fig. 1), is one of
the biggest commercial clam beds in Korea. It is characterized by a
well-developed sand-mud tidal flat. For the analysis, 30-40 clams were
collected monthly in 1999 and bimonthly in 2000. On arrival at the
laboratory, clams were kept in a seawater tank for 24 h to depurate
sediments in the stomach. Shell length (i.e., longest axis of the shell)
and the wet tissue weight of each clam were recorded. Clams with a shell
length more than 27 mm (i.e., age, >2 y) were included exclusively in
the analysis because Perkinsus infection in the Manila clam is often
size dependent (Park & Choi 2001, Villalba et al. 2004). The
condition index (CI) was calculated as a ratio of wet tissue weight to
shell length (Park & Choi 2004).

[FIGURE 1 OMITTED]

To determine the infection intensity, 10 mL fluid thioglycollate
medium (FTM) was placed in a 15-mL plastic conical tube and sterilized.
After cooling, 200 U mycostatin (nystatin) and 2 mg chloromycetin
(chloramphenicol) dissolved in 50 [micro]L distilled water was added to
each tube to depress bacterial growth during incubation. Whole tissue of
each clam was then added to the test tube and incubated at room
temperature (20-25[degrees]C) for a week in the dark (Ray 1952, Ray
1966). After incubation, the tubes were centrifuged at 760g for 10 min
to discard FTM. Ten milliliters 2-M NaOH was then added to each tube and
incubated at 50[degrees]C for 2 h to digest the tissue (Choi et al.
1989). The digested clam tissues were washed several times with
phosphate-buffered saline (pH 7.3) by centrifuging at 800g for 10 min.
The total number of P. olseni cells in each clam was then determined by
counting the prezoosporangia in a 1/10 or 1/100 diluted clam subsample
using a hemocytometer. The infection intensity was then expressed as the
number of P. olseni cells per gram wet tissue.

To test seasonality in the infection intensity, the sampling months
were grouped as spring (March, April, and May), summer (June, July, and
August), fall (September, October, and November), and winter (December,
January, and February). Analysis of variance (ANOVA) with Duncan's
multiple range test was performed using SAS statistical analysis
software to test the seasonality, and the test was run by year to
determine interannual variation.

RESULTS

A total of 510 clams with a shell length that ranged from 30.0
(August 1999)-36.6 mm (July 1999), and a wet tissue weight of 1.052
(August 1999)-2.988 g (July 1999) were used in the analysis (Table 1).
The monthly mean CI ranged from 3.435 (October 2000)-7.809 (July 1999;
Fig. 2). CI increased gradually from February to July, then decreased
dramatically in August in 1999 as a result of a massive clam spawning
during this period.

Monthly prevalence (i.e., percentage of infected clam) of P. olseni
infection in Manila clam ranged from 83 (April 2000)-100%, with a mean
of 97%. The prevalence was remarkably high (100%) during most of the
sampling periods. Because of the high prevalence, no clear seasonal
pattern was observed in the prevalence during the 2 y of sampling.

[FIGURE 2 OMITTED]

Figure 3 shows seasonal changes in infection intensity, which
ranged from 366,001 (July 1999)-2,235,325 P. olseni cells/g tissue
(October 1999). ANOVA with Duncan's multiple range test showed
that, in 1999, the infection intensity recorded in fall and spring was
significantly higher than the value recorded in spring and summer (P
< 0.001). In year 2000, the infection intensity measured in fall was
significantly higher compared with other seasons, whereas P. olseni
cells per gram tissue recorded in summer was significantly lower than
the value recorded in any other season (P < 0.001; Table 2).

DISCUSSION

There was no clear seasonality in P. olseni prevalence in Gomso
Bays as a result of the exceptionally high prevalence year-round, as was
also reported in previous studies. From March 1999 to February 2000,
Park et al. (2006a) investigated monthly prevalence and infection
intensity of P. olseni in the Manila clam in Gomso Bay using RFTM and
the 2-M NaOH digestion technique. In their study, the prevalence and
infection intensity were assessed from the gill tissue, because other
parts of the tissues were used in other biochemical analysis. Of the 14
mo of sampling, the monthly prevalence was 100% for 13 sampling periods,
except December 1999 (70%) (Park et al. 2006a). Leite et al. (2004) also
reported no obvious seasonal variation of P. olseni infection prevalence
in the carpet shell clam Ruditapes decussatus along the Portuguese
coast, where prevalence ranged from 20-100%. In contrast, Villalba et
al. (2005) observed a clear seasonality in prevalence: high in spring
and summer when the surface water temperature remained warm. Recently,
Uddin et al. (2010) also observed a seasonal difference in the
prevalence of P. olseni in the Manila clam in Incheon Bay, approximately
200 km to the north of Gomso Bay. In Incheon Bay, prevalence ranged from
38-97% annually, and was significantly higher in fall than in spring and
summer, when most clam are in the postspawning stage.

[FIGURE 3 OMITTED]

Contrary to prevalence, infection intensity did show a clear
seasonality. The infection intensity recorded in 1999 did show a
different seasonal pattern, and the intensity recorded during the fall
and winter was significantly greater than the values recorded during the
spring and summer. In 2000, the different seasonal pattern was observed
again, with infection intensity highest in the fall and lowest during
the summer. It is remarkable that the infection intensity increased
dramatically from August (486,827 cells/g tissue) to September
(1,254,707 cells/g tissue) in 1999 (Fig. 3). Such a dramatic increase in
intensity could be attributable to the warn: water temperatures during
August and September, and to the poor physiological condition of the
clams. The CI of clams in the fall is much lower than spring and early
summer, and this difference is a result of the spawning activity of the
clam (Fig. 2). According to Park and Choi (2004), the Manila clam in
Gomso Bay spawns from late May to September, with a strong spawning
pulse in late July. In the fall, most clams are spent reproductively or
are in a resting phase (Park & Choi 2004). Several studies have
reported mass mortalities or poor health conditions in marine bivalves
during postspawning periods (Cheney et al. 2000, Costil et al. 2005,
Royer et al. 2008). Using flow cytometry, Hong (2010) first investigated
the immunological condition of the Manila clam inhabiting the west coast
of Korea during the postspawning period. In October 2010, Manila clams
collected from Goheung, off the south coast of Korea, were mostly spent
(60%) or partially spawned (25%). Flow cytometry results indicated that
hemocyte viability and the phagocytosis activity of clams that were
spent or in the resting stage were poorer than those of clams in the
ripe (or prespawning) stage. Therefore, it is believed that the dramatic
increase of P. olseni infection intensity in the Manila clam from late
summer to fall in Gomso Bay could be associated with the poor
immunological condition of the clams during postspawning period, which
might enhance proliferation of the parasites in clam bodies. It is also
postulated that the extremely high number of P. olseni observed during
the fall in Gomso Bay could be responsible for the autumn mass mortality
of clams recurring in Gomso Bay.

The recurring mortality of clams during the fall in Gomso Bay is
believed to be the result of complex interactions among the
physiological conditions of the clam, P. olseni infection, and the
environmental conditions in the bay. Quantity and quality of food
available to the clan: have drawn little attention in relation to clam
parasite interactions, although several studies have demonstrated that
the food condition modulates a defense-related mechanism in the clam
(Chu & La Peyre 1993, Chu et al. 1993, Hegaret et al. 2004,
Delaporte et al. 2003, Delaporte et al. 2006a, Delaporte et al. 2006b,
Delaporte et al. 2007). Choi et al. (1989) first estimated the energy
consumption of Perkinsus marinus in the Eastern oyster in Galveston Bay
and concluded that P. marinus in heavily infected oysters requires more
energy than the oyster would have available after meeting its own
metabolic demand. Casas (2002) also estimated the energy demand of P.
olseni in the market-sized carpet shell clam R. decussatus. In Galicia
Spain, the energy demand of the high level of P. olseni infection in the
carpet shell clam exceeded the energy available to the clam for its own
growth, especially under conditions of warm temperatures and low food
availability. The negative energy balance caused by the progression of
Perkinsus infection in the hosts could help to explain the lethal and
sublethal effects (Villalba et al. 2004).

Depending on the season, the effects of an extremely high level of
P. olseni infection observed during the spring and fall could reflect
differently on clam physiology. According to Park and Choi (2004), there
were late spring to early summer and fall chlorophyll a peaks (9-11
[micro]g/L) in Gomso Bay in 1999. Compared with spring and early summer,
the chlorophyll levels were low (24 [micro]g/L) during late fall and
winter 1999 and 2000. During spring and early summer 1999, Manila clams
could have overcome physiological stresses caused by the heavy
infection, because the food availability was high enough to compensate
for the energetic cost of the infection. However, during late fall to
winter, when food availability is relatively low, P. olseni would
probably reclaim most of the available energy, inducing mortality of the
most heavily infected clams. It is also likely that the spawning
activities of clams during summer and early fall in the bay exhausted
the clams, which may aggravate the defense capability of the clan.
Consequently, the combined effects of the low level of food supply and
the clam's poor physiological condition may facilitate the
proliferation of P. olseni in clam tissues during fall and winter.

In summary, infection intensity of P. olseni in the adult Manila
clam in Gomso Bay, off the west coast of Korea, was monitored for 2 y
(1999 and 2000) using RFTM and the 2-M NaOH digestion technique. Monthly
prevalence of P. olseni was mostly 100%, except for a few sampling
months, and no clear seasonality was observed in prevalence. Infection
intensity was significantly greater in fall and winter, when food
availability in the bay was considered to be poor. It is believed that
seasonal change in the infection intensity was governed externally by
seasonal changes in water temperature and salinity, and internally by
the annual reproductive cycle of the clam.

ACKNOWLEDGMENT

We are grateful to the staff members of the Shellfish Aquaculture
and Research Laboratory at Jeju National University for their support in
data acquisition. This study was supported by the research grant of Jeju
National University (2008-2009) to K. S. C.

(1) School of Marine Biomedical Science (POST BK-21) and Marine and
Environmental Research Institute, Jeju National University, 66
Jejudaehakno, Jeju 690-756, Republic of Korea; (2) East Sea Environment
Research Department, East Sea Branch of Korea Ocean Research and
Development Institute, Uljin, Republic of Korea; (3) Department of
Aquatic Life Medicine, Kunsan National University, Kunsan, Republic of
Korea